WO2015157546A1 - Biomarqueurs protéiques pour évaluation immunitaire et prédiction de rejet de greffe - Google Patents

Biomarqueurs protéiques pour évaluation immunitaire et prédiction de rejet de greffe Download PDF

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WO2015157546A1
WO2015157546A1 PCT/US2015/025164 US2015025164W WO2015157546A1 WO 2015157546 A1 WO2015157546 A1 WO 2015157546A1 US 2015025164 W US2015025164 W US 2015025164W WO 2015157546 A1 WO2015157546 A1 WO 2015157546A1
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aact
apoe
crp
saa
a1at
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PCT/US2015/025164
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Elaine F. REED
Hans Albin GRITSCH
Jeffrey Lorne VEALE
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The Regents Of The University Of California
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Priority to CA2944990A priority Critical patent/CA2944990A1/fr
Priority to KR1020167031144A priority patent/KR20160142390A/ko
Priority to AU2015243424A priority patent/AU2015243424A1/en
Priority to US15/302,463 priority patent/US10451636B2/en
Priority to EP15777119.7A priority patent/EP3129781A4/fr
Priority to JP2016561603A priority patent/JP2017513009A/ja
Publication of WO2015157546A1 publication Critical patent/WO2015157546A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates generally to detection, prediction, and monitoring of transplant rejection, including renal allograft rejection.
  • the invention more specifically pertains to use of protein markers that can be detected in serum for early defection of allograft rejection.
  • a transplant to replace damaged tissues with healthy ones from either living or decreased individuals of the same species is called an allograft.
  • Tissues and cells from another individual are usually recognized as foreign by the mammalian immune system. This stimulates a response of the host defenses to reject the foreign tissue.
  • the immune system can occasionally recognize very small differences in molecular structure and develop an alloimmune response.
  • Some cell types are very similar among individuals such as blood cells. It is possible to administer blood products such red blood ceils, and plasma without an immune response if the blood type is compatible. Virtually all mammalian ceils, except red blood cells, express major histocompatibility molecules on their cell surface. These molecules are a critical mechanism by which the immune system can distinguish self from foreign cells.
  • Organ transplantation is the definitive therapy for many forms of end-stage disease of the kidney, liver, heart, lung, pancreas and intestine. Transplantation of specialized cells such as islets of Langerhans and nerve cells are becoming more common, as well as complex tissue transplantation such as a limb or face.
  • Transplantation of specialized cells such as islets of Langerhans and nerve cells are becoming more common, as well as complex tissue transplantation such as a limb or face.
  • the long-term success of these procedures is limited by the immune response.
  • the donor is an identical twin, most recipients must take immunosuppressive medications.
  • Current immunosuppressive therapies are not capable of preventing rejection in all cases, and have multiple significant side effects. These drugs may lead to life threatening infection, cardiovascular disease, diabetes, and cancer.
  • Chronic transplant dysfunction is a phenomenon in solid organ transplants with a gradual deterioration of function accompanied by characteristic histological features on graft biopsy. In kidney transplantation, this is known as chronic rejection, chronic allograft nephropathy (CAN), or interstitial fibrosis with tubular atrophy (IFTA). In heart transplantation there is accelerated atherosclerosis and in liver transplantation the bile ducts atrophy. Chronic transplant injury is characterized by fibrosis of the internal blood vessels of the transplant and may be related to sub-clinical AMR. Detecting this problem at an early stage is difficult even with protocol biopsy.
  • the invention provides a set of protein markers and methods of using these markers for assessment of a patient's immune status and for predicting rejection of an organ transplant.
  • the markers include complement C4 anaphy!atoxin (C4A), Apolipoprotein A1 (ApoA1 ), a-1 anti-chymotrypsin C terminal fragment (AACT), C1 protease inhibitor (C-inh), Serum Amyloid A (SAA), C-reactive Protein (CRP), Apolipoprotein E (ApoE). aipha-1 -antitrypsin (A1AT), Post- transiationai modified ApoA1 , Inter-aipha-trypsin inhibitor heavy chain H4 (ITIH4). and Alpha-2- macrogiobulin (A2M), as well fragments of these proteins (see Tables 1 and 4, and SEQ ID NOs: 1 -18).
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection, which can be achieved with a high degree of sensitivity and specificity.
  • the method detects rejection with at least 90% sensitivity and at least 90% specificity
  • the method comprises the steps of: (a) measuring the amount of a 5 kiloDaiton (kDa) fragment of complement C4 anaphyiatoxin (C4A) in a sample obtained from the patient; (b) comparing the amount of the 5 kDa fragment of C4A in the patient sample with the amount in a control sample; and (c) detecting susceptibility to graft rejection when the comparing shows an increase in the 5 kDa fragment of C4A in the patient sample relative to the control sample
  • the method comprises the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds a 5 kiloDaiton (kDa) fragment of complement C
  • the contacting further comprises contacting the patient sample with a reagent that binds Apollpoprofein A1 (ApoA1 ) and/or a-1 anti-chymotrypsln C terminal fragment (AACT), and the detecting further comprises detecting susceptibility to graft rejection when the comparing shows a decrease in the amount of ApoAI and/or AACT in the patient sample relative to the control sample.
  • the contacting and comparing of (b) and (c) comprise contacting the patient sample with reagents that bind ApoAI and AACT.
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection, in one embodiment, the method comprises the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds a set of biomarkers comprising two or more markers listed in Table 1 ; (b) measuring the amount of specific binding between the reagents and the patient sample; (c) comparing the amount of specific binding in (b) with the amount of specific binding of the reagents to a control sample; and (d) detecting susceptibility to graft rejection when the comparing in (c) shows a significant increase or decrease in specific binding to the biomarkers in the patient sample relative to the control sample, in some embodiments, steps (a)-(c) are performed for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, up to 19 of the markers listed in Table 1.
  • the set of markers consists of 8 or fewer markers listed in Table 1 . In another embodiment, the set of markers consists of 6 or fewer markers listed in Table 1 . In yet another embodiment, the set of markers consists of 4 or fewer markers listed in Table 1 .
  • susceptibility to graft rejection is detected when the specific binding of reagent to marker is increased in the patient sample relative to the control: C1 protease inhibitor (d -inh), Serum Amyloid A (SAA), C-reactive Protein (CRP), Apo!ipoprotein E (ApoE), alpha-1 -antitrypsin (A1AT), and/or specific binding of reagent to the following markers is decreased relative to control: AACT, ApoAl , Post-translationai modified ApoAl , Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Aipha-2-macrogiobuiin (A2M).
  • the difference is a decrease or increase of at least 10% relative to the control.
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection, the method comprising the steps of: (a) contacting a sample obtained from the patient with reagents that specifically bind a set of markers consisting of: a 5191 Dalton fragment of complement C4 anaphylatoxin (C4A), a-1 anti-chymotrypsin C terminal fragment (AACT), and Apo A1 ; and optionally further consisting of: complement C1 inhibitor, and/or a marker listed in Table 1 ; (b) measuring the amount of specific binding between the reagent and the sample; (c) comparing the amount of specific binding in (b) with a control sample; and (d) detecting susceptibility to graft rejection when the comparing in (c) shows an increase in the 5191 Dalton fragment of C4A or a decrease AACT and/or ApoAl relative to the control sample.
  • C4A complement C4 anaphylatoxin
  • AACT -1 anti-chymotryp
  • the measuring comprises an immunoassay, in other words
  • the measuring comprises mass spectrometry.
  • reagents include, but are not limited to, an antibody, a nucleic acid probe, or a synthetic probe.
  • the probe or antibody may optionally be labeled with a detectable marker.
  • solid organ grafts include, but are not limited to, kidney, liver, heart, pancreas, lung, intestine and thymus.
  • the solid organ graft rejection is acute cellular renal allograft rejection.
  • Other types of solid organ graft rejection include chronic rejection, such as chronic allograft nephropathy, or interstitial fibrosis with tubular atrophy.
  • the invention additionally provides a kit comprising antibodies that specifically bind the 5 kDa fragment of C4A, ApoAl , AACT, and, optionally, one or more additional markers listed in Table 1.
  • the kit comprises reagents that bind C4A (or a 5 kDa fragment thereof) and ApoAl (or the fragment thereof at amino acids 148-183) and/or AACT (or the fragment thereof at amino acids 385-422), in one embodiment, the kit comprises reagents that specifically bind the 5191 Da fragment of AACT and ApoAl .
  • the kit further comprises a solid support onto which the antibodies are immobi!ized.
  • a solid support examples include, but are not limited to, a microtiter plate, beads, a membrane or other support known to those skilled in the art.
  • the antibodies are immobilized via binding to antigen that is immobilized to the solid support.
  • the antibodies are immobilized via binding to a bead or particle such as luminex.
  • the kit further comprises a chromogenic substrate.
  • a method for detecting susceptibility in a patient to solid organ graft rejection comprising the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds complement C4 anaphylatoxin (C4A); (b) measuring the amount of specific binding between the reagent and the sample; (c) comparing the amount of specific binding in (b) with a control sample; wherein a greater amount of binding in (b) relative to the control is indicative of susceptibility to solid organ graft rejection.
  • C4A complement C4 anaphylatoxin
  • Another illustrative embodiment of the invention is a mass spectrometry assay to identify the peptide and/or protein profile in a patient that is associated with solid organ allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or one of the other 18 proteins in Table 1 in plasma or serum or other body fluid, the assay comprising the steps of: (a) measuring the specific amount of specific peptide by mass spectrometry in plasma, serum or other body fluid; (b) comparing the specific quantity of protein/peptide in (a) with a control sample; wherein a greater or lesser amount of specific peptide relative to the control is indicative of susceptibility to solid organ graft rejection.
  • kits to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor in plasma or serum, the kit comprising: (a) a microtiter piate coated with polyclonal or monoclonal antibodies specific to a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (c) p- nitropheny!-phosphate; and (d) a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1
  • a method for detecting susceptibility in a patient to acute cellular renal allograft rejection comprising the steps of: (a) providing polyclonal or monoclonal antibodies against a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (b) providing a microtiter piate coated with the antibodies; (c) adding the serum or plasma to the microtiter piate; (d) providing alkaline phosphatase-antibody conjugates reactive with ⁇ -1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor to the microtiter plate; (e) providing p-nitrophenyl-phosphate to the microtiter plate; and (f) comparing the reaction which occurs as a result of steps (a) to (e) with a standard curve to determine the levels of a- 1 anti-chymotrypsin and/or Apo A1 and/or
  • kits to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific to a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (c) p- nitrophenyi-phosphate; and (d) a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor
  • kits to screen in plasma, serum and ⁇ or biological fluid for a molecular profile in a patient that is associated with solid organ allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or proteins described in Table 1 the kit comprising: (a) a microbead array coated with polyclonal or monoclonal antibodies specific to a-1 anti- chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or other proteins listed in Table 1 ; (b) polyclonal or monoclonal antibody fluorescent dye conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or other proteins listed in Table 1 ; (c) and a-1 anti-chymotrypsin and/or Apo A1 and/or
  • Embodiments of the invention provide assays and methods for the detection and/or quantitation in a sample of: AACT and/or Apo A1 and/or C4A and/or SAA and/or CRP and/or A2M and/or ApoE and/or ITIH4 and/or A1 AT and/or C-inh and/or fragments of these polypeptides.
  • Typical embodiments of the invention utilize ELISA-type assays of the type that are suitable for use with biological fluid samples such as blood, plasma, serum, or other bodily fluids of a mammal, particularly a human.
  • Methodological embodiments include testing for the amounts of AACT and/or C4A and/or Apo A1 polypeptides, fragments of these markers, and/or a combination of the markers described herein in biological fluid samples.
  • embodiments examine together: a -1 antichymotrypsin, Apo A1 , complement C1 inhibitor and the 5191 Da peptide of C4A in order to, for example, identify a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection.
  • Certain embodiments examine 1 (e.g. the 5191 Da C4A peptide) or 2 (e.g. C4A peptide plus a -1 antichymotrypsin or Apo A1 ) or 3 (e.g. a -1 antichymotrypsin, Apo A1 and the 5191 Da C4A peptide) or ail 4 of these biomarkers.
  • the 5191 Da C4A peptide is purified and this purified peptide is then injected into animals such as a rabbits to generate polyclonal antibodies specific to the 5191 Da peptide that can be used in ELISA tests or the like.
  • animals such as a rabbits to generate polyclonal antibodies specific to the 5191 Da peptide that can be used in ELISA tests or the like.
  • monoclonal antibody preparations to the protein may be prepared by injecting the purified 5191 Da peptide into mice, harvesting the spleen and lymph node cells, fusing these cells with mouse myeloma cells and using the resultant hybridoma cells to produce the monoclonal antibody.
  • One illustrative embodiment of the invention is a method for detecting a-1 antichymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor in plasma to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection comprising the steps of: (a) providing polyclonal or monoclonal antibodies against a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor; (b) providing a microtiter plate coated with the antibodies; (c) adding the serum or plasma to the microtiter plate; (d) providing alkaline phosphatase-antibody conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor to the microtiter plate; (e) providing p-nitrophenyi-phosphate
  • kits to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor in plasma or serum
  • the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific to a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor; (c) p-nitrophenyl- phosphate; and (d) a-1 anti-chymotrypsin and/
  • Figs. 1 A-1 C Candidate plasma proteins which detect renal allograft rejection.
  • Figs. 2A-2D identification of the C-termina! fragment of ⁇ -1 antichymotrypsin and apolipoprotein A1 (Apo A1 ).
  • Figs. 3A-3D Validation of apolipoprotein A1 (Apo A1 ) by enzyme-linked immunosorbent assay (ELISA) for association with acute cellular rejection.
  • ELISA enzyme-linked immunosorbent assay
  • 3A Box plots of the ELISA results of plasma samples of a subset of the original cohort.
  • 3B Box plots of the ELISA results of the plasma samples of the second independent cohort.
  • 3C Box plots of Apo A1 levels in recipients that had a sample collected within - 3 to 0 days before the renal biopsy. Twenty-five rejection and 22 nonrejection samples were compared. The mean Apo A1 levels in nonrejection was 0.22 SD ⁇ 0.06 and 0.13 SD ⁇ 0.04 in rejection.
  • Fig. 4 ApoA1 comparison between biopsy proven non-rejectors versus biopsy proven rejectors, showing that patients diagnosed with renal allograft rejection had significantly lower Apo A1 levels (mean 0.156+/- 0.77) compared to non-rejection (mean 0.239+/-0.16)
  • Fig. 5 Sensitivity and specificity of ApoA1 comparison between biopsy proven non- rejectors versus biopsy proven rejectors. Using a outpoint of 0.195, Apo A1 levels correctly classified patients with rejection 79% of the time with a sensitivity of 75% and specificity of 82%.
  • Fig. 8 Validation of Alpha-2-macroglobuiin using an antibody-based ELISA.
  • the invention described herein is based on the discovery that specific protein markers present in human plasma can be used to detect, predict and monitor transplant rejection.
  • sample from a subject means a specimen obtained from the subject that contains plasma, blood, serum, saliva, urine, or other bodily fluid.
  • the term "subject” includes any human or non-human animal.
  • the term “non-human animal” includes ail vertebrates, e.g., mammals and non-mammals, such as non- human primates, horses, sheep, dogs, cows, pigs, chickens, amphibians, reptiles, rodents etc.
  • the subject, or patient is a human.
  • a "control" sample means a sample that is representative of normal measures of the respective marker, or a baseline amount of marker to be used for comparison.
  • the baseline will be a measurement taken from the same subject or patient.
  • the sample can be an actual sample used for testing, or a reference level or range, based on known normal measurements of the corresponding marker.
  • 5 kiioDalton (kDa) fragment of complement C4 anaphylatoxin (C4A) refers to a fragment of C4A that is approximately 5 kDa in molecular weight, and includes a 5,051 Da C4A fragment that corresponds to SEQ ID NO: 3, and a 5,191 Da C4A fragment that corresponds to SEQ ID NO: 2.
  • the 5 kDa fragment of C4A is the 5,191 Da fragment.
  • the invention provides a set of protein markers and methods of using these markers for assessment of a patient's immune status and for predicting rejection of an organ transplant.
  • the markers include complement C4 anaphylatoxin (C4A), Multeipoprotein A1 (ApoA1 ), a-1 anti-chymotrypsin C terminal fragment (AACT), C1 protease inhibitor (C-inh), Serum Amyloid A (SAA), C-reactive Protein (CRP), aceaipoprotein E (ApoE), alpha-1 -antitrypsin (A1AT), Post- transiationai modified ApoA1 , inter-aipha-trypsin inhibitor heavy chain H4 (ITIH4), and Alpha-2- macrogiobulin (A2M), as well fragments of these proteins (see Tables 1 and 4, and SEQ ID NOs: 1-18). Changes in the levels of these markers in a patient's plasma or other bodily fluid are predictive of allograft rejection.
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection.
  • the method defects susceptibility to rejection with greater than 80% sensitivity and specificity, and in a typical embodiment, with at least 90% sensitivity and at least 90% specificity.
  • the method comprises the steps of: (a) measuring the amount of a 5 kiioDalton (kDa) fragment of complement C4 anaphyiatoxin (C4A) in a sample obtained from the patient; (b) comparing the amount of the 5 kDa fragment of C4A in the patient sample with the amount in a control sample; and (c) detecting susceptibility to graft rejection when the comparing shows an increase in the 5 kDa fragment of C4A in the patient sample relative to the control sample
  • the method comprises the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds a 5 kiioDalton (kDa) fragment of complement C4 anaphyiatoxin (C4A); (b) measuring the amount of specific binding between the reagent and the patient sample; (c) comparing the amount of specific binding in (b) with the amount of specific binding of the reagent in a control sample; and (d)
  • the contacting further comprises contacting the patient sample with a reagent that binds AINipoprotein A1 (ApoA1 ) and/or a-1 anti-chymotrypsin C terminal fragment (AACT), and the detecting further comprises detecting susceptibility to graft rejection when the comparing shows a decrease in the amount of ApoAI and/or AACT in the patient sample relative to the control sample, in one embodiment, the contacting and comparing comprise contacting the patient sample with reagents that bind ApoAI and AACT.
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection, in one embodiment, the method comprises the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds a set of biomarkers comprising two or more markers listed in Table 1 ; (b) measuring the amount of specific binding between the reagents and the patient sample; (c) comparing the amount of specific binding in (b) with the amount of specific binding of the reagents in a control sample; and (d) detecting susceptibility to graft rejection when the comparing in (c) shows a significant increase or decrease in specific binding to the biomarkers in the patient sample relative to the control sample, in some embodiments, steps (a)-(c) are performed for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, up to 19 of the markers listed in Table 1.
  • the set of markers consists of 8 or fewer markers listed in Table 1 . In another embodiment, the set of markers consists of 6 or fewer markers listed in Table 1 . In yet another embodiment, the set of markers consists of 4 or fewer markers listed in Table 1 .
  • susceptibility to graft rejection is detected when the specific binding of reagent to marker is increased relative to the control: C4A, C1 protease inhibitor (C1-inh), Serum Amyloid A (SAA), C-reactive Protein (CRP), aceaipoprotein E (ApoE), aipha-1 -antitrypsin (A1AT), including fragments thereof, and/or specific binding of reagent to the following markers is decreased relative to control: AACT, ApoA1 , Post-translational modified ApoA1 , inter-aipha-trypsin inhibitor heavy chain H4 (ITIH4), Alpha-2-macrogiobuiin (A2M), including fragments thereof, in a typical embodiment, the difference is a decrease or increase of at least 10% relative to the control.
  • C1-inh C1 protease inhibitor
  • SAA Serum Amyloid A
  • CRP C-reactive Protein
  • ApoE aipha-1 -antitrypsin
  • the invention provides a method for detecting susceptibility in a patient to solid organ graft rejection, the method comprising the steps of: (a) contacting a sample obtained from the patient with reagents that specifically bind a set of markers consisting of: a 5191 Dalton fragment of complement C4 anaphylatoxin (C4A), a-1 anti-chymotrypsin C terminal fragment (AACT), and Apo A1 ; and optionally further consisting of: complement C1 inhibitor, and/or a marker listed in Table 1 ; (b) measuring the amount of specific binding between the reagent and the sample; (c) comparing the amount of specific binding in (b) with the amount of specific binding of the reagent to a control sample; and (d) detecting a set of markers consisting of: a 5191 Dalton fragment of complement C4 anaphylatoxin (C4A), a-1 anti-chymotrypsin C terminal fragment (AACT), and Apo A1 ; and optionally further consisting of
  • the measuring comprises an immunoassay, in other words
  • the measuring comprises mass spectrometry.
  • Other assay methods include fluorescence activated cell sorting (FACS), western blotting, and amplification of a surrogate DNA template.
  • Representative examples of reagents include, but are not limited to, an antibody, a nucleic acid probe, or a synthetic probe.
  • the probe or antibody may optionally be labeled with a detectable marker, in some embodiments, the reagents are labeled with a detectable marker and/or observed using a chromogenic or fluorogenic substrate.
  • solid organ grafts include, but are not limited to, kidney, liver, heart, pancreas, lung, intestine and thymus.
  • the solid organ graft rejection is acute cellular renal allograft rejection.
  • Other types of solid organ graft rejection include chronic rejection, such as chronic allograft nephropathy, or interstitial fibrosis with tubular atrophy.
  • a method for detecting susceptibility in a patient to solid organ graft rejection comprising the steps of: (a) contacting a sample obtained from the patient with a reagent that specifically binds complement C4 anaphylatoxin (C4A); (b) measuring the amount of specific binding between the reagent and the sample; (c) comparing the amount of specific binding in (b) with a control sample; wherein a greater amount of binding in (b) relative to the control is indicative of susceptibility to solid organ graft rejection.
  • C4A complement C4 anaphylatoxin
  • Another illustrative embodiment of the invention is a mass spectrometry assay to identify the peptide and/or protein profile in a patient that is associated with solid organ allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or one of the other 18 proteins in Table 1 in plasma or serum or other body fluid, the assay comprising the steps of: (a) measuring the specific amount of specific peptide by mass spectrometry in plasma, serum or other body fluid; (b) comparing the specific quantity of protein/peptide in (a) with a control sample; wherein a greater or lesser amount of specific peptide relative to the control is indicative of susceptibility to solid organ graft rejection.
  • a method for detecting susceptibility in a patient to acute cellular renal allograft rejection comprising the steps of: (a) providing polyclonal or monoclonal antibodies against a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (b) providing a microtiter plate coated with the antibodies; (c) adding the serum or plasma to the microtiter plate; (d) providing alkaline phosphatase-antibody conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor to the microtiter plate; (e) providing p-nitropheny!-phosphate to the microtiter plate; and (f) comparing the reaction which occurs as a result of steps (a) to (e) with a standard curve to determine the levels of a-1 anti-chymotrypsin and/or Apo A1 and/or C4A
  • Embodiments of the invention provide assays and methods for the detection and/or quantitation in a sample of: AACT and/or Apo A1 and/or C4A and/or SAA and/or CRP and/or A2M and/or ApoE and/or ITIH4 and/or A1 AT and/or C-inh and/or fragments of these polypeptides.
  • Typical embodiments of the invention utilize ELISA-type assays of the type that are suitable for use with biological fluid samples such as blood, plasma, serum, or other bodily fluids of a mammal, particularly a human.
  • Methodological embodiments include testing for the amounts of AACT and/or C4A and/or Apo A1 polypeptides, fragments of these markers, and/or a combination of the markers described herein in biological fluid samples.
  • Certain embodiments examine together: a -1 antichymotrypsin, Apo A1 , complement C1 inhibitor and the 5191 Da peptide of C4A in order to, for example, identify a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection.
  • Certain embodiments examine 1 (e.g. the 5191 Da C4A peptide) or 2 (e.g. C4A peptide plus a -1 antichymotrypsin or Apo A1 ) or 3 (e.g. a -1 antichymotrypsin, Apo A1 and the 5191 Da C4A peptide) or ail 4 of these biomarkers.
  • the 5191 Da C4A peptide is purified and this purified peptide is then injected into animals such as a rabbits to generate polyclonal antibodies specific to the 5191 Da peptide that can be used in ELISA tests or the like.
  • animals such as a rabbits to generate polyclonal antibodies specific to the 5191 Da peptide that can be used in ELISA tests or the like.
  • monoclonal antibody preparations to the protein may be prepared by injecting the purified 5191 Da peptide into mice, harvesting the spleen and lymph node ceils, fusing these ceils with mouse myeloma ceils and using the resultant hybridoma cells to produce the monoclonal antibody.
  • One illustrative embodiment of the invention is a method for detecting a-1 anti- chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor in plasma to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection comprising the steps of: (a) providing polyclonal or monoclonal antibodies against a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor; (b) providing a microtiter plate coated with the antibodies; (c) adding the serum or plasma to the microtiter plate; (d) providing alkaline phosphatase-antibody conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor to the microtiter plate; (e) providing p-nitrophenyi-
  • an amount of marker is considered increased or decreased if it differs by a statistically significant amount from the amount present in the control, in some embodiments, the difference is an increase or decrease of at least 10%; in other embodiments, the difference is at least 20%, 30%, 40%, 50% or more.
  • a reference range has been identified for the amount of the marker present in a normal, control sample, and a test sample having an amount of the marker that is outside the reference range for that marker is susceptible to rejection.
  • the sample is a plasma sample.
  • Other bodily fluids can be used for the sample, including serum, urine and saliva.
  • markers for which an increase relative to control is indicative of rejection include C4A, CRP, SAA, C1 -inh, A1AT, and ApoE.
  • Markers for which a decrease relative to control is indicative of rejection include AACT, ApoA1 , ITIH4, and A2M.
  • the measuring comprises chromatography or spectrometry.
  • the chromatography can be gas or liquid chromatography.
  • the spectrometry can be mass spectrometry.
  • Other known methods of marker detection are also contemplated, and may be selected based on the characteristics of the individual marker of interest. Examples of other assays that can be employed include immunoassay and electrochemical detection. Measures of test samples can be compared directly to controls, such as comparing the amount of the marker present in the test sample to the amount of the marker in a control sample or to a known normal level of the marker. Alternatively, in some embodiments, the marker amount is compared to a baseline amount for the same subject.
  • WO 2007/13801 1 US 2007/0202085; US 7,235,358; US 2007/0037166; US
  • kits are also within the scope of the invention.
  • kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the containers ) comprising one of the separate elements to be used in the method.
  • the probes, antibodies and other reagents of the kit may be provided in any suitable form, including frozen, lyophi!ized, or in a pharmaceutically acceptable buffer such as TBS or PBS.
  • the kit may also include other reagents required for utilization of the reagents in vitro or in vivo such as buffers (i.e., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and / or detection reagents (i.e., goat anti-mouse IgG biotin, streptavidin ⁇ HRP conjugates, allophycocyanin, B-phycoerythrin, R- phycoerythrin, peroxidase, fluors (i.e., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HILyte Fluor 647), and / or staining kits (I.e., ABC Staining Kit, Pierce)).
  • buffers i.e., TBS, PBS
  • blocking agents solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein
  • kits may also include other reagents and / or instructions for using antibodies, probes, and other reagents in commonly utilized assays described above such as, for example, liquid or gas chromatography, spectrometry, electrochemical assay, flow cytometric analysis, ELISA, immunoblotting (i.e., western blot), immunocytochemistry, immunohistochemistry.
  • the kit provides the reagent in purified form, in another
  • the reagents are immunoreagents that are provided in biotinyiated form either alone or along with an avidin-conjugated detection reagent (i.e., antibody).
  • the kit includes a fluorescent!y labeled immunoreagent which may be used to directly detect antigen. Buffers and the like required for using any of these systems are well- known in the art and may be prepared by the end-user or provided as a component of the kit.
  • the kit may also include a solid support containing positive- and negative-control protein and / or tissue samples.
  • kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS-PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a label can be provided on the container to indicate that the composition is used for a specific application, and can also indicate directions for use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
  • the invention additionally provides a kit comprising antibodies that specifically bind the 5 kDa fragment of C4A, ApoAl , AACT, and, optionally, one or more additional markers listed in Table 1.
  • the kit comprises reagents that bind C4A (or a 5 kDa fragment thereof) and ApoAl (or the fragment thereof at amino acids 148-183) and/or AACT (or the fragment thereof at amino acids 385-422), in one embodiment, the kit comprises reagents that specifically bind the 5191 Da fragment of AACT and ApoAl .
  • the kit further comprises a solid support onto which the antibodies are immobilized.
  • a solid support examples include, but are not limited to, a microtiter plate, beads, a membrane or other support known to those skilled in the art.
  • the antibodies are immobilized via binding to antigen that is immobilized to the solid support.
  • the antibodies are immobilized via binding to a bead or particle such as luminex.
  • the kit further comprises a chromogenic substrate.
  • kits to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disciosed herein and/or complement C1 inhibitor in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific to a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disclosed herein and/or complement C1 inhibitor; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or the 5191 peptide disciosed herein and/or complement C1 inhibitor; (c) p-nitropheny!- phosphate; and (d) a-1 anti-chymotiter plate coated with polyclonal or monoclonal antibodies specific to
  • kits to screen for a plasma molecular profile in a patient that is associated with acute cellular renal allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific to a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or compiement C1 inhibitor; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor; (c) p ⁇ nitrophenyi-phosphate; and (d) a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C
  • kits to screen in plasma, serum and ⁇ or biological fluid for a molecular profile in a patient that is associated with solid organ allograft rejection by detecting a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or proteins described in Table 1 the kit comprising: (a) a microbead array coated with polyclonal or monoclonal antibodies specific to a-1 anti- chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or other proteins listed in Table 1 ; (b) polyclonal or monoclonal antibody fluorescent dye conjugates reactive with a-1 anti-chymotrypsin and/or Apo A1 and/or C4A and/or complement C1 inhibitor and/or other proteins listed in Table 1 ; (c) and a-1 anti-chymotrypsin and/or Apo A1 and/or
  • Peptides 5191 Da and 4467 Da detected rejection with 100% sensitivity and 94% specificity.
  • Table 2 illustrates the demographics of the study population and shows no significant differences with respect to age, gender, race, transplant number, or degree of human leukocyte antigen mismatch. The percentage of deceased and living donors was similar to U.S. Organ Procurement Transplant Network national data.
  • ATG antithymog!obulin
  • IL2Ra inter!eukin-2 receptor alpha chain
  • SD standard deviation
  • HLA human leukocyte antigen
  • Ail biopsies were performed for cause and taken after a serum creatinine rise or delayed graft function. Two of the ACR positive biopsies also had evidence of AMR, whereas four of the biopsies had evidence of onlyAMR.
  • 20 of 27 were collected within 3 months after the biopsy, 3 of 27 were within 6 months, 1 of 27 was collected at 9 months, and 3 of 27 were collected 1 year after the biopsy.
  • 38 of 51 were collected within 3 months after the biopsy, 5 of 51 were within 8 months, 4 of 51 were collected within 9 months after the biopsy, and 4 of 51 were collected after 1 year.
  • Ail biopsies from this group of nonrejectors were performed for cause and taken after a serum creatinine rise or delayed graft function.
  • maintenance immunosuppression included a calcineurin inhibitor (tacrolimus or cyclosporine), an antiproliferative agent (mycophenolate mofetil or sirolimus), and prednisone. In some cases without evidence of pretransplant sensitization, steroids were withdrawn.
  • ProteinChip arrays (CM10 and Q10) were preequilibrated for binding. pH fractions were bound to the arrays and analyzed in triplicate. Six pooled human serum (Sigma, St. Louis, MO) samples were used as internal controls on each array set. After sample array processing, matrix was applied: either saturated sinapinic acid (SPA, LaserBio Labs, Sophia-Antipoiis, France) or 20% saturated alpha-cyano-4-hydroxy cinnamic acid (CHCA, LaserBio Labs). A Biomek 2000 liquid handling robot (Beckman, Fuiierton, CA) was used for matrix spotting.
  • SPA saturated sinapinic acid
  • CHCA LaserBio Labs
  • a Biomek 2000 liquid handling robot (Beckman, Fuiierton, CA) was used for matrix spotting.
  • ProteinChip Software 3.2 was calibrated in the high mass range before reading the SPA- spotted arrays (laser energies of 150 and 170). A peptide mixture was used for calibration of the CHCA-spotted arrays (laser energies of 120 and 135). Mass spectrum from each spot was an average of 240 laser shots. All spectra were preliminarily analyzed In CiphergenExpress Client Software 3.0 to create clusters by Expression Difference Mapping analysis. A cluster is a group of peaks with the same mass/charge (m/z) ratio and treated as the same protein or peptide across multiple spectra. Peak intensities from each spectrum were used to measure relative protein/peptide amounts. Total ion current was used to normalize the signals. The reproducibility of peak heights was similar to what was previously published (37), showing a CV of 15% to 30%.
  • MALDI-TOF- S was performed with a prOTOF 2000 orthoganoi-TOF mass
  • Nonparametric (binomial) and parametric (mixed effects and linear regression) models were used to identify significant peaks. Two-sided P values less than 0.05 were considered significant, and Bonferroni correction was used as needed. CART analysis was conducted to select a subset of candidate biomarkers and determine their ability to correctly classify rejection. Adjusted intensities of the selected protein/peptide peaks were used as the predictor variables in CART training. The R software package (version 3.1 , at r-project.org) was used for statistical computations. Traditional logistic regression analysis was performed, and ROC plots were generated using STATA data analysis and statistical software. Cut points were selected by the maximum correctly classified. Details of the statistical analyses are presented in the Supplemental material (see Supplemental Digital Content 1 , links.lww.com/TP/A465;
  • the number refers to the pH fraction.
  • the “Ratio” indicates whether the protein intensity is higher in rejection (A) or postrejection (B) patients.
  • C aIpha-cyano-4-hydroxy cinnarnic acid
  • S sinapinic acid
  • L low laser energy
  • H high laser energy
  • m/z mass to charge ratio
  • the pattern of Apo A1 levels measured by ELISA (Fig. 3A) is consistent with the SELDI intensity peak levels (Fig. 2D).
  • the Apo A1 ELISA was performed on an independent cohort consisting of 27 plasma samples from patients with biopsy-confirmed acute cellular rejection (ACR) with a corresponding postrejection sample and 51 patients with biopsy-proven nonrejection.
  • the prerejection samples demonstrated Apo A1 levels nearly identical to the average levels of the postrejection samples (Fig. 3D).
  • the Apo A1 medial level was 0.14 for all three groups indicating that the severity of rejection was not distinguished by the Apo A1 level.
  • AACT and Apo A1 desorption/ionization time-of-fiight mass spectrometry were significantly associated with the diagnosis of acute allograft rejection.
  • the identity of two of these proteins, AACT and Apo A1 was elucidated.
  • the C-terminal fragment of AACT is significantly lower during rejection compared with postrejection and nonrejection.
  • a different variation of the AACT fragment (4354 Da) is found at higher levels in urine of patients with acute rejection compared with those with stable transplants (14).
  • Pimenta et al. (17) examined the hydrolysis of AACT by cathepsin D using high-performance liquid chromatography isolation and MADLI and found three peptides: 4354 Da, 4468 Da, and 4625 Da. These cleavage products are identical to the 4467 Da peak identified in plasma and the 4354 Da found in urine based on the sequence data, suggesting that both may be produced by cathepsin D.
  • Apo A1 and synthetic peptides which mimic Apo A1 activity, have strong antiinflammatory and antioxidant properties (22, 23).
  • the Apo A1 mimetic peptide, D-4F reduces intimal lesions caused by chronic rejection in a mouse model. The mechanism of this effect involves induction of the antioxidant gene heme oxygenase-1 in the graft and/or a direct effect on T-lymphocyte proliferation and effector cytokine production (24).
  • isolation and identification of the 5191 Da peptide have been hindered due to its low abundance in plasma, its copurification with other more abundant peptides, and the inability to directly fragment it. For unknown reasons, some peptides fragment poorly and yield MS/MS spectra that cannot be deciphered (33, 34).
  • a combination of the 5191 and 4467 Da peaks robustly assessed rejection verses nonrejection, whereas the combination of the 4467 and 28 kDa peaks was not as valuable, thus identification of the 5191 Da peak is important.
  • a limitation of our study design is that the plasma was collected at routine patient visits, which was not always on the same day that the renal biopsy was performed. Also, the clinical parameters of our study population were not uniform with respect to immunosuppression. Furthermore, only two patients in the original cohort had AMR, and therefore, a signature for AMR could not be determined. A majority of patients rejected within the first 100 days, and thus, we were limited to only explore early rejection, in the future, it will be also valuable to explore these markers in patients undergoing late renal allograft rejection. Additionally, we did not find an association between severity of rejection based on Banff classification and Apo A1 levels. It would be of interest to attempt this comparison again in a larger cohort of subjects.
  • Example 1 The 28 kDa protein identified in Example 1 as Apo-A1 consistently showed lower levels in patients with biopsy proven renal allograft rejection.
  • ELISA enzyme-linked immunosorbent assay
  • the aim of the current study was to validate the predictive value of the marker for renal aiiograft rejection using an independent cohort of 73 renal aiiograft recipients transplanted at UCLA between 2010 and 2013. 45/71 patients had rejection while 28 patients had biopsy proven non-rejection, [0100] Plasma samples from 73 renal recipients were assayed for Apo- A1 protein levels by ELISA. 45 of the patients experienced at least one biopsy proven rejection episode while 28 patients had biopsy proven no-rejection. Plasma samples obtained within 7 days prior or 2 days after diagnosis of rejection were quantitated by ELISA for Apo A1 levels. Renal allograft rejection was diagnosed using Banff criteria.
  • Figure 6 presents ELISA data showing the same pattern of detection of A!pha-2- macrogiobu!in as was demonstrated using mass spectrometry. Paired plasma samples were collected at the time of rejection and post-rejection from 7 renal allograft recipients diagnosed with rejection. These are the same paired serum samples that were used in Example 1 above. The data show that the levels of Aipha-2-macrogiobu!in as detected by ELISA decrease at the time of diagnosis of rejection.
  • Alpha-1 -antitrypsin C-Terminal fragment (2505 Da, A1AT 397-418; SEQ ID NO: 1 1 ) LMIEQNTKSPLFMGKWNPTQK
  • Apolipoprotein A1 fragment (4125 Da, Apo A1 148-183; SEQ ID NO: 5)
  • Plasma protease C1 inhibitor fragment (4187 Da, C1 inh 445-478; SEQ ID NO: 13)
  • Alpha-1 -antichymotrypsin C-terminal fragment (4467 Da, AACT 385-422; SEQ ID NO: 8)
  • Serum Amyloid A-4 (1 1694 Da, SAA4 21-122; SEQ ID NO: 14)
  • C-reactive protein (19409 Da, CRP 10-185; SEQ ID NO: 15)
  • Apolipoprotein A1 (28077 Da, Apo A1 58-267; SEQ ID NO: 6)
  • Apolipoprotein E (36747 Da, Apo E 19-317, Glycosylated; SEQ ID NO: 16)

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Abstract

L'invention concerne un procédé de dépistage et de détection d'un rejet de greffe d'organe solide chez un sujet qui comprend l'analyse d'un échantillon de patient de plasma, de sérum ou de sang provenant du sujet pour une protéine marqueuse identifiée dans la description. Une quantité élevée ou réduite de marqueur présent dans l'échantillon du patient par rapport à un échantillon témoin est indicatrice d'un rejet, et identifie des sujets nécessitant une biopsie ou un traitement modifié. Le procédé peut être utilisé pour cribler des patients présentant un risque de rejet de greffe sans devoir subir des procédures de biopsie plus coûteuses, risquées et invasives.
PCT/US2015/025164 2014-04-09 2015-04-09 Biomarqueurs protéiques pour évaluation immunitaire et prédiction de rejet de greffe WO2015157546A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA2944990A CA2944990A1 (fr) 2014-04-09 2015-04-09 Biomarqueurs proteiques pour evaluation immunitaire et prediction de rejet de greffe
KR1020167031144A KR20160142390A (ko) 2014-04-09 2015-04-09 면역평가 및 이식거부의 예측을 위한 단백질 바이오마커
AU2015243424A AU2015243424A1 (en) 2014-04-09 2015-04-09 Protein biomarkers for immune assessment and prediction of transplant rejection
US15/302,463 US10451636B2 (en) 2014-04-09 2015-04-09 Protein biomarkers for immune assessment and prediction of transplant rejection
EP15777119.7A EP3129781A4 (fr) 2014-04-09 2015-04-09 Biomarqueurs protéiques pour évaluation immunitaire et prédiction de rejet de greffe
JP2016561603A JP2017513009A (ja) 2014-04-09 2015-04-09 免疫評価および移植片拒絶の予測のためのタンパク質バイオマーカー

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US201461977567P 2014-04-09 2014-04-09
US61/977,567 2014-04-09

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US (1) US10451636B2 (fr)
EP (1) EP3129781A4 (fr)
JP (1) JP2017513009A (fr)
KR (1) KR20160142390A (fr)
AU (1) AU2015243424A1 (fr)
CA (1) CA2944990A1 (fr)
WO (1) WO2015157546A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019129824A1 (fr) * 2017-12-27 2019-07-04 Vito Nv Biomarqueurs de typage de receveurs d'allogreffe
WO2023212285A1 (fr) * 2022-04-28 2023-11-02 Children's Hospital Medical Center Procédés de prédiction et de traitement d'un dysfonctionnement chronique de l'allogreffe pulmonaire

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210238681A1 (en) * 2018-03-12 2021-08-05 The Regents Of The University Of California Assessing Transplant Rejection Status by Analysis of T-Cell Receptor Subunit Repertoire Diversity
CN112375138B (zh) * 2020-11-13 2022-12-13 中元汇吉生物技术股份有限公司 一种重组载脂蛋白e和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053195A1 (en) * 2004-09-06 2009-02-26 Friedrich Raulf Biomarkers for acute graft rejection
US8003333B2 (en) * 2007-09-28 2011-08-23 Mayo Foundation For Medical Education And Research Serum biomarkers for early detection of acute cellular rejection

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104371A1 (en) 1997-09-24 2003-06-05 Strom Terry B. Methods of evaluating transplant rejection
GB0018614D0 (en) 2000-07-28 2000-09-13 Rose Marlene L Organ transplant rejection and associated conditions
US7026121B1 (en) 2001-06-08 2006-04-11 Expression Diagnostics, Inc. Methods and compositions for diagnosing and monitoring transplant rejection
US7235358B2 (en) 2001-06-08 2007-06-26 Expression Diagnostics, Inc. Methods and compositions for diagnosing and monitoring transplant rejection
AU2003281287A1 (en) 2002-07-04 2004-01-23 Oxford Glycosciences (Uk) Ltd Toxicity markers
AU2003275363A1 (en) 2002-10-01 2004-04-23 The Johns Hopkins University Use of biomarkers for detecting acute renal transplant rejection
WO2006029184A2 (fr) 2004-09-08 2006-03-16 Expression Diagnostics, Inc. Genes permettant de diagnostiquer et de surveiller les troubles associes a l'inflammation
US7666596B2 (en) 2005-05-23 2010-02-23 University Of Alberta Tissue rejection
EP1993573A2 (fr) 2006-01-13 2008-11-26 Renovar, Inc. Systemes et procedes de controle de reponses immunitaires et prediction de resultats chez des porteurs de greffe
GB0605217D0 (en) 2006-03-15 2006-04-26 Novartis Ag Method and compositions for assessing acute rejection
GB0607943D0 (en) 2006-04-21 2006-05-31 Novartis Ag Biomarkers for chronic transplant dysfunction
WO2007138011A1 (fr) 2006-05-25 2007-12-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés de diagnostic et de traitement d'un rejet de greffe et de pathologies inflammatoires
WO2008084331A2 (fr) 2006-06-21 2008-07-17 Hopitaux Universitaires De Geneve Biomarqeurs pour des troubles rénaux
US7993832B2 (en) 2006-08-14 2011-08-09 Xdx, Inc. Methods and compositions for diagnosing and monitoring the status of transplant rejection and immune disorders
CA2661487A1 (fr) 2006-08-28 2008-03-06 Source Precision Medicine Etablissement de profil d'expression genique pour l'identification, la surveillance et le traitement d'un rejet de greffon
GB0617429D0 (en) 2006-09-05 2006-10-18 Electrophoretics Ltd Markers of renal transplant rejection and renal damage
WO2009039421A1 (fr) 2007-09-20 2009-03-26 University Of Louisville Research Foundation, Inc. Biomarqueurs peptidiques prédictifs d'un déclin de la fonction rénale et d'une maladie des reins
WO2009045104A1 (fr) 2007-10-05 2009-04-09 Academisch Ziekenhuis Bij De Universiteit Van Amsterdam Procédé de diagnostic d'un rejet présymptomatique de greffe d'organes
EP2088205A1 (fr) 2008-02-11 2009-08-12 Institut National De La Sante Et De La Recherche Medicale (Inserm) PSMB10 : marqueur de diagnostic et cible thérapeutique de rejet chronique
WO2009114699A2 (fr) 2008-03-12 2009-09-17 The Trustees Of Columbia University In The City Of New York Ngal à poids moléculaire élevé en tant que biomarqueur pour une maladie rénale chronique
EP2131200A1 (fr) 2008-06-04 2009-12-09 BRAHMS Aktiengesellschaft Marqueur pour le rejet de greffe et la mortalité
ES2434996T3 (es) 2008-10-31 2013-12-18 St Vincent's Hospital Sydney Limited Métodos de pronóstico en enfermedad renal crónica
EP2368113A1 (fr) 2008-12-19 2011-09-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Biomarqueurs pour le diagnostic d'une allogreffe rénale et de l'état des reins
US20110065136A1 (en) 2009-08-07 2011-03-17 Rules-Based Medicine, Inc. Methods and Devices for Detecting Glomerulonephritis and Associated Disorders
MX339765B (es) 2010-02-05 2016-06-08 Astute Medical Inc Metodos y composiciones para diagnostico y pronostico de lesion renal y falla renal.
EP3761034A3 (fr) 2011-01-26 2021-02-17 University of Pittsburgh - Of the Commonwealth System of Higher Education Biomarqueurs d'urine pour la prédiction de la récupération après une lésion rénale aiguë : protéomique
EP2518512A1 (fr) 2011-04-29 2012-10-31 Medizinische Universität Wien Méthode pour indiquer une maladie rénale chez un patient en déterminant au moins une protéine de HDL isolées
US20130052665A1 (en) * 2011-08-25 2013-02-28 Bruce Xuefeng Ling Methods for diagnosis of systemic juvenile idiopathic arthritis
US20140038203A1 (en) 2012-07-09 2014-02-06 Musc Foundation For Research Development Methods for detecting or predicting kidney disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053195A1 (en) * 2004-09-06 2009-02-26 Friedrich Raulf Biomarkers for acute graft rejection
US8003333B2 (en) * 2007-09-28 2011-08-23 Mayo Foundation For Medical Education And Research Serum biomarkers for early detection of acute cellular rejection

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BAO, YANG ET AL.: "Proteomic profiling of heterotopic heart-transplanted rats using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry: Potential biomarkers and drug targets", JOURNAL OF INTERNATIONAL MEDICAL RESEARCH, vol. 41, no. 3, 2013, pages 628 - 635, XP055230247, ISSN: 0300-0605 *
LIN, DAVID CHIA-HSIANG: "Biomarkers of acute and chronic human heart allograft rejection", DOCTORAL DISSERTATION, THE UNIVERSITY OF BRITISH COLUMBIA, PATHOLOGY AND LABORATORY MEDICINE, 2012, XP055230244 *
ROEDDER, SILKE ET AL.: "Biomarkers in solid organ transplantation: establishing personalized transplantation medicine", GENOME MEDICINE, vol. 3, no. 6, 2011, pages 1 - 12, XP055173790 *
See also references of EP3129781A4 *
ZIEGLER, MARY E. ET AL.: "Apolipoprotein A1 and C-terminal fragment of alpha-1 antichymotrypsin are candidate plasma biomarkers associated with acute renal allograft rejection", TRANSPLANTATION, AUTHOR MANUSCRIPT, vol. 92, no. Issue 4, 2011, pages 1 - 15, XP055230243, ISSN: 0041-1337 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019129824A1 (fr) * 2017-12-27 2019-07-04 Vito Nv Biomarqueurs de typage de receveurs d'allogreffe
US20200400685A1 (en) * 2017-12-27 2020-12-24 Vito Nv Biomarkers for Typing Allograft Recipients
WO2023212285A1 (fr) * 2022-04-28 2023-11-02 Children's Hospital Medical Center Procédés de prédiction et de traitement d'un dysfonctionnement chronique de l'allogreffe pulmonaire

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CA2944990A1 (fr) 2015-10-15
US10451636B2 (en) 2019-10-22
EP3129781A1 (fr) 2017-02-15
KR20160142390A (ko) 2016-12-12
EP3129781A4 (fr) 2017-10-11
US20170030928A1 (en) 2017-02-02
AU2015243424A1 (en) 2016-10-27
JP2017513009A (ja) 2017-05-25

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